Cotargeting a MYC/eIF4A-survival axis improves the efficacy of KRAS inhibitors in lung cancer.

Despite the success of KRAS G12C inhibitors in non-small cell lung cancer (NSCLC), more effective treatments are needed. One preclinical strategy has been to cotarget RAS and mTOR pathways; however, toxicity due to broad mTOR inhibition has limited its utility. Therefore, we sought to develop a more refined means of targeting cap-dependent translation and identifying the most therapeutically important eukaryotic initiation factor 4F complex-translated (eIF4F-translated) targets. Here, we show that an eIF4A inhibitor, which targets a component of eIF4F, dramatically enhances the effects of KRAS G12C inhibitors in NSCLCs and together these agents induce potent tumor regression in vivo. By screening a broad panel of eIF4F targets, we show that this cooperativity is driven by effects on BCL-2 family proteins. Moreover, because multiple BCL-2 family members are concomitantly suppressed, these agents are broadly efficacious in NSCLCs, irrespective of their dependency on MCL1, BCL-xL, or BCL-2, which is known to be heterogeneous. Finally, we show that MYC overexpression confers sensitivity to this combination because it creates a dependency on eIF4A for BCL-2 family protein expression. Together, these studies identify a promising therapeutic strategy for KRAS-mutant NSCLCs, demonstrate that BCL-2 proteins are the key mediators of the therapeutic response in this tumor type, and uncover a predictive biomarker of sensitivity.

Combating castration-resistant prostate cancer by co-targeting the epigenetic regulators EZH2 and HDAC.

While screening and early detection have reduced mortality from prostate cancer, castration-resistant disease (CRPC) is still incurable. Here, we report that combined EZH2/HDAC inhibitors potently kill CRPCs and cause dramatic tumor regression in aggressive human and mouse CRPC models. Notably, EZH2 and HDAC both transmit transcriptional repressive signals: regulating histone H3 methylation and histone deacetylation, respectively. Accordingly, we show that suppression of both EZH2 and HDAC are required to derepress/induce a subset of EZH2 targets, by promoting the sequential demethylation and acetylation of histone H3. Moreover, we find that the induction of one of these targets, ATF3, which is a broad stress response gene, is critical for the therapeutic response. Importantly, in human tumors, low ATF3 levels are associated with decreased survival. Moreover, EZH2- and ATF3-mediated transcriptional programs inversely correlate and are most highly/lowly expressed in advanced disease. Together, these studies identify a promising therapeutic strategy for CRPC and suggest that these two major epigenetic regulators buffer prostate cancers from a lethal response to cellular stresses, thereby conferring a tractable therapeutic vulnerability.

Coordinating gene expression during the cell cycle.

Cell cycle-dependent gene transcription is tightly controlled by the retinoblastoma (RB):E2F and DREAM complexes, which repress all cell cycle genes during quiescence. Cyclin-dependent kinase (CDK) phosphorylation of RB and DREAM allows for the expression of two gene sets. The first set of genes, with peak expression in G1/S, is activated by E2F transcription factors (TFs) and is required for DNA synthesis. The second set, with maximum expression during G2/M, is required for mitosis and is coordinated by the MuvB complex, together with B-MYB and Forkhead box M1 (FOXM1). In this review, we summarize the key findings that established the distinct control mechanisms regulating G1/S and G2/M gene expression in mammals and discuss recent advances in the understanding of the temporal control of these genes.

MMB-FOXM1-driven premature mitosis is required for CHK1 inhibitor sensitivity.

To identify genes whose loss confers resistance to CHK1 inhibitors, we perform genome-wide CRISPR-Cas9 screens in non-small-cell lung cancer (NSCLC) cell lines treated with the CHK1 inhibitor prexasertib (CHK1i). Five of the top six hits of the screens, MYBL2 (B-MYB), LIN54, FOXM1, cyclin A2 (CCNA2), and CDC25B, are cell-cycle-regulated genes that contribute to entry into mitosis. Knockout of MMB-FOXM1 complex components LIN54 and FOXM1 reduce CHK1i-induced DNA replication stress markers and premature mitosis during Late S phase. Activation of a feedback loop between the MMB-FOXM1 complex and CDK1 is required for CHK1i-induced premature mitosis in Late S phase and subsequent replication catastrophe, indicating that dysregulation of the S to M transition is necessary for CHK1 inhibitor sensitivity. These findings provide mechanistic insights into small molecule inhibitors currently studied in clinical trials and provide rationale for combination therapies.

STRIPAK directs PP2A activity toward MAP4K4 to promote oncogenic transformation of human cells.

Alterations involving serine-threonine phosphatase PP2A subunits occur in a range of human cancers, and partial loss of PP2A function contributes to cell transformation. Displacement of regulatory B subunits by the SV40 Small T antigen (ST) or mutation/deletion of PP2A subunits alters the abundance and types of PP2A complexes in cells, leading to transformation. Here, we show that ST not only displaces common PP2A B subunits but also promotes A-C subunit interactions with alternative B subunits (B''', striatins) that are components of the Striatin-interacting phosphatase and kinase (STRIPAK) complex. We found that STRN4, a member of STRIPAK, is associated with ST and is required for ST-PP2A-induced cell transformation. ST recruitment of STRIPAK facilitates PP2A-mediated dephosphorylation of MAP4K4 and induces cell transformation through the activation of the Hippo pathway effector YAP1. These observations identify an unanticipated role of MAP4K4 in transformation and show that the STRIPAK complex regulates PP2A specificity and activity.

RB, p130 and p107 differentially repress G1/S and G2/M genes after p53 activation.

Cell cycle gene expression occurs in two waves. The G1/S genes encode factors required for DNA synthesis and the G2/M genes contribute to mitosis. The Retinoblastoma protein (RB) and DREAM complex (DP, RB-like, E2F4 and MuvB) cooperate to repress all cell cycle genes during G1 and inhibit entry into the cell cycle. DNA damage activates p53 leading to increased levels of p21 and inhibition of cell cycle progression. Whether the G1/S and G2/M genes are differentially repressed by RB and the RB-like proteins p130 and p107 in response to DNA damage is not known. We performed gene expression profiling of primary human fibroblasts upon DNA damage and assessed the effects on G1/S and G2/M genes. Upon p53 activation, p130 and RB cooperated to repress the G1/S genes. In addition, in the absence of RB and p130, p107 contributed to repression of G1/S genes. In contrast, G2/M genes were repressed by p130 and p107 after p53 activation. Furthermore, repression of G2/M genes by p107 and p130 led to reduced entry into mitosis. Our data demonstrates specific roles for RB, p130-DREAM, and p107-DREAM in p53 and p21 mediated repression of cell cycle genes.

Cyclin D-CDK4 relieves cooperative repression of proliferation and cell cycle gene expression by DREAM and RB.

The retinoblastoma protein (RB) restricts cell cycle gene expression and entry into the cell cycle. The RB-related protein p130 forms the DREAM (DP, RB-like, E2F, and MuvB) complex and contributes to repression of cell cycle-dependent genes during quiescence. Although both RB and DREAM bind and repress an overlapping set of E2F-dependent gene promoters, it remains unclear whether they cooperate to restrict cell cycle entry. To test the specific contributions of RB and DREAM, we generated RB and p130 knockout cells in primary human fibroblasts. Knockout of both p130 and RB yielded higher levels of cell cycle gene expression in G0 and G1 cells compared to cells with knockout of RB alone, indicating a role for DREAM and RB in repression of cell cycle genes. We observed that RB had a dominant role in E2F-dependent gene repression during mid to late G1 while DREAM activity was more prominent during G0 and early G1. Cyclin D-Cyclin-Dependent Kinase 4 (CDK4)-dependent phosphorylation of p130 occurred during early G1, and led to the release of p130 and MuvB from E2F4 and decreased p130 and MuvB binding to cell cycle promoters. Specific inhibition of CDK4 activity by palbociclib blocked DREAM complex disassembly during cell cycle entry. In addition, sensitivity to CDK4 inhibition was dependent on RB and an intact DREAM complex in both normal cells as well as in palbociclib-sensitive cancer cell lines. Although RB knockout cells were partially resistant to CDK4 inhibition, RB and p130 double knockout cells were significantly more resistant to palbociclib treatment. These results indicate that DREAM cooperates with RB in repressing E2F-dependent gene expression and cell cycle entry and supports a role for DREAM as a therapeutic target in cancer.